Since the advent of double-stranded RNA inhibition (RNAi) as a tool for controlling gene expression, as described in WO 99/32619 and WO 00/01846, there has been recognized a need for specialized vectors designed for the production of double-stranded RNA (dsRNA).
Cloning vectors designed to produce high levels of dsRNA have been previously described by Plaetinck et al. (WO 00/01846) and Timmons et al. Nature, 395:854 (1998). These vectors generally contain a multiple cloning site (MCS) into which target DNA fragments can be cloned flanked by two opposable transcriptional promoters. Essentially, these three components (Promoter 1, MCS and Promoter 2) make up the entire system. In the appropriate expression system, the DNA cloned into the MCS may be transcribed in both directions, leading to the production of two complementary RNA strands.
A disadvantage of the known systems is that not only the cloned fragment is transcribed. Read-through of the RNA polymerase will result in transcription of the entire vector, and this also in both directions. As only transcription of the cloned DNA fragment will result in active dsRNA for RNAi purposes, transcription of the vector part results in useless, inefficient RNA. More specifically, 80% of these transcripts can be considered as non-specific and thus non-effective.
The large amounts of non-specific RNA generated by the prior art plasmid and expression systems results in some undesirable side effects. First, in RNAi protocols based on introduction of dsRNA into C. elegans via a food organism such as E. coli which expresses the dsRNA (see WO 00/01846), large RNA strands are considered to be toxic for the food organism. As a result, high amounts of RNA accumulating in E. coli cause a significant part of the population to die.
Second, and probably more important, is the reduction of inhibition potential. The presence of large amounts of non-specific dsRNA causes a competitive environment for the specified sequences. The potential of the template-specified dsRNA sequences to inhibit the targeted protein expression in, for instance, C. elegans cells is reduced by the presence of these large non-specific regions. Such an inhibition by non-specific dsRNA has also been shown in Drosophila by Tushl et al., Genes & Development 13:3191-3197 (1999). Not only the potential to inhibit gene expression is affected, but also the amount of specific dsRNA produced is limited.
Third, transcription of the vector backbone part, more particularly transcription of the origin of replication and related structures, results in plasmid instability and plasmid reorganisation, leading to reduced production of dsRNA. This relatively low concentration of effective dsRNA in turn leads to inefficient RNAi.
To conclude, the previously described vectors have following shortcomings: they are toxic to the feeding organism, a greater proportion of the transcripts produced are non-specific, the inhibitory potential of the dsRNA is reduced by the presence of non-specific regions, a high incidence of plasmid reorganizations and loss of plasmid from the feeding organism. It is therefore an object of the present invention to provide improved vectors for the production of dsRNA which avoid the disadvantages of the prior art vectors.
Vectors for use in the in vitro synthesis of RNA transcripts, for example the production of RNA probes, have been known and commonly used in the art for some time (see for example F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); Jendrisak et al, Vectors for in vitro production of RNA copies of either strand of a cloned DNA sequence, U.S. Pat. No. 4,766,072). In standard in vitro transcription protocols the problem of read-through transcription of vector sequences is generally avoided by linearizing the transcription vector at restriction site positioned at the 3′ end of the desired transcript. However, this solution is not appropriate for in vivo transcription or for the production of dsRNA where it is important that the template is transcribed in both directions.